(699d) High Throughput Platform for Macromolecular Transport

The expansion of mAb treatments available resulted in a need for advancement in administration routes. Subcutaneous administration is less invasive; requires shorter clinic times, which results in a significant improvement in patient’s quality of life; improves patient compliance; and reduces cost to the healthcare system. The injected biotherapeutic, however, needs to traverse complex structures of the subcutis and the extracellular matrix (ECM) before it is absorbed by the lymphatic system to have an active effect. The ECM is largely composed of collagen, a fibrous protein, and hyaluronic acid (HA), and anionic polysaccharide. The high viscosity of HA is believed to act as a barrier for therapeutic transport. Transwell macromolecular recovery assay is a promising method to study transport of macromolecules through biological barriers. The Transwell inserts are permeable support devices which utilize permeable membrane which allows transport of macromolecules while trapping larger components (such as cells and fibrillar components of the extracellular matrix). In this work, we study the transport of a panel of macromolecules through tissue models composed of collagen and HA. Set of molecules was chosen to include Lysozyme (Lys, a small positive molecule), beta-lactoglobulin (BLg, a negative molecule), Dextran at 20 kDa (Dex, a linear molecule), bovine serum albumin (BSA; large negative molecule), and Bovine IgG (a large molecule which resembles therapeutic monoclonal antibodies in size).

Recovery of five model molecules through collagen at 4 mg/mL (Col4) were performed demonstrated collagen matrix acts as size sorter. In these curves, the ranking for recovery after 48 hours is Lys>BLg~Dex>BSA>IgG. This ranking is directly related to the Stokes radii of the molecules, with Lys being the smallest molecule at 1.9 nm, followed by BLg at 3.5 nm, Dex at 3.2 nm, BSA at 4.1 nm and IgG at 5 nm. As control, recovery through HA at 20 mg/mL (1500 kDa) was tested. The ranking for recovery at 48 hours changed to BSA>BLg>IgG>Dex>Lys. BSA is the most negative molecule tested, thus the matrix composed of solely HA (a negative polysaccharide) likely repels BSA, and thus increased recovery is observed. Lysozyme on the other hand is the most positive molecule, which likely experiences attractive electrostatic forces which slow down its recovery. To compare the contributions of both collagen and HA simultaneously, hydrogel composed of collagen at 4 mg/mL and HA at 2 mg/L (Col4HA2) was tested. The ranking for recovery after 48 hours in Col4HA2 is Lys>BLg>Dex~BSA>IgG. These results are similar to the results in Col4 hydrogels, however, the recovery appears to be inhibited by the presence of HA.

Collagen and HA play pivotal role in macromolecular transport. Collagen provides a tortuosity barrier while HA inhibits to the transport through its viscous properties. While free HA matrix recovery is governed primarily by electrostatic forces, recovery in ColHA matrices is a function of mainly the Col microstructure, meanwhile HA inhibits recovery.